Since the elucidation of the structure in 1971 of the luteinizing hormone releasing hormone (LHRH), the international chemical, biological and clinical research on this hormone has been enormous. A category of analogs which are more active than LHRH evolved from various investigators, but all have a D-amino acid in place of Gly.sup.6, and have been known as "superagonists". Multiple citations to all this chemistry on LHRH are in "Advances in Reproductive Health Care, LHRH and Its Analogs Contraceptive and Therapeutic Applications", edited by B. H. Vickery, J. J. Nestor Jr., and E. S. E. Hafez, MTP Press Limited, (1984).
Retaining the L-configuration for each of the ten positions of LHRH, the literature (LHRH, listed by NICHD, Mar. 16, 1983), records more than 100 monosubstituted, and about 14 disubstituted analogs, all from naturally occurring amino acids, which have agonist activity, but apparently not one of these 114 analogs has been found to have agonist activity as potent as that of LHRH. Of these 114 agonist-analogs, [Ala.sup.9 ]-LHRH (LHRH, listed by NICHD, Mar. 16, 1983, No. 247) showed 65% activity in vitro, and [Phe.sup.5 ]-LHRH (Coy, D. H., Coy, E. J., Schally, A. V., J. Med. Chem. 16, 83 (1973)) showed 64% activity in vivo. [Trp.sup.7 ]-LHRH (E) was once found to have 80% activity in vivo, (Cone, B. S. et al., Biochem. Biophys. Res. Commun. 90, 1249 (1979)).
Two analogs, both of which contain an unnatural amino acid, were apparently equal to and better than LHRH, in vitro. These two analogs were [3(2-Nal).sup.3 ]-LHRH (LHRH, listed by NICHD, Mar. 16, 1983, No. 82) and [N-MeLeu.sup.7 ]-LHRH (Ling, N., Vale, W., Biochem. Biophys. Res. Commun. 63, 801 (1975)), which were 200% and 100% as active as LHRH, in vitro, respectively,
Apparently there has been no achievement of an agonist comparable to LHRH, i.e., equivalent potency for release of both LH and FSH, without substitutions with unnatural or the D-forms of natural amino acids.
The existence of a follicle stimulating hormone releasing hormone (FSHRH) has been both proposed and denied, and is still unresolved. Igarashi, M. and McCann, S. M., (Endocrinology 74, 446-452 (1964)) described the initial studies on a presumed FSHRH. Schwartz summarized physiological evidence which indicated that there must be some separate secretory control of FSH, (Folkers, K., Fuchs, S., Humphries, J., Wan, Y. P., and Bowers, C. Y., The Follicle Stimulating Hormone Releasing Hormone in Novel Aspects of Reproductive Physiology, Spilman, C. H., and Wilks, J. W., eds., Spectrum Publicatios, Inc., New York, 336 (1978)). The initial denials against FSHRH were made in 1970-71 by White, W. F., (Hypophysiotropic Hormones of the Hypothalamus, J. Meites, ed., The Williams and Wilkins Co., Baltimore, p. 248 (1970)), and by Schally, A. V., Arimura, A., Baba, Y., Nair, R. M. G., Matsuo, H., Redding, T. W., Debeljuk, L. and White, W. F., (Biochem. Biophys. Res. Commun. 43, 393 (1971)) and again by Matsuo, H., Baba, Y., Nair, R. M. G., Arimura, A. and Schally, A. V., ( Biochem. Biophys. Res. Commun. 43, 1334 (1971)), and they stated--"one polypeptide regulates secretion of luteinizing and follicle stimulating hormones"--and they introduced the designation "gonadotropin releasing hormone" (GnRH), which has been widely used in endocrinology. Koch, Y., et al. (Biochem. Biophys. Res. Commun. 55, 623-629 (1973)) and Wise, P. M., et al. (Endocrinology 104, 940-947 (1979)) described results interpreted on the basis that LHRH is also FSHRH.
Several investigators described data on purification and bioassays in support of the existance of FSHRH. In 1973, Johanssen, K. N. G. et al., (Biochem. Biophys. Res. Commun. 50, 8-13 (1973)), Curry, B. L., et al. (Biochem. Biophys. Res. Commun. 50, 14-19 (1973)) and Bowers, C. Y., et al. (Biochem. Biophys. Res. Commun. 50, 20-26 (1973)) described fractions which released 40,000-128,000 ng/ml of FSH in comparison to synthetic LHRH which released ca. 18,000 ng/ml. Isarashi, M., et al. (Psychoneuroendocrinology. Workshop Conf. Int. Soc. Phychoneuroendocrinology, Mieken, Karger, Basel, 178-186 (1974)), Fawcett, C. P., et al. (Endocrinology 96, 1311-1314 (1975)), Yu, J. Y. L., et al. (Life Sciences 22, 269-282 (1978)), Yu, J. Y. L., et al. (Neuroendocrinology 29, 54-65 (1979)), and Blask, D. E., et al. (Neuroendocrinology 28, 36-43 (1979)), have all published results supporting the existance of FSHRH.
Fuchs, S., et al. (Biochem. Biophys. Res. Commun. 88, 92-96 (1979)) purified an entity, which unambiguously released FSH, was separated from [.sup.3 H]-LHRH and which appeared to have a molecular weight larger than that of LHRH, and which seemed not to be a prohormone of LHRH. Igarashi et al. (Kitakanto Igaku. 32(5) 409-421 (1982)), published a review of 29 publications on the existance of FSHRH as distinct from LHRH in porcine hypothalamus.
Concerning prohormonal forms of LHRH, Millar, R. P., et al. (Colloq. Int. C.N.R.S., 487-510 (1978)), Millar, R. P., et al. (Biochem. Biophys. Res. Commun. 74, 720-731 (1977)), and King, J. A. and Millar, R. P., (Science 206, 67-69 (1979)) described important results.
Four naturally occuring LHRH's are now known as follows.
__________________________________________________________________________ 12345678910 __________________________________________________________________________ Porcine/Ovine: pGlu--His--Trp--Ser--Tyr--Gly--Leu--Arg--Pro--Gly--NH.sub.2 Salmon: pGlu--His--Trp--Ser--Tyr--Gly--Trp--Leu--Pro--Gly--NH.sub.2 Chicken I: pGlu--His--Trp--Ser--Tyr--Gly--Leu--Gln--Pro--Gly--NH.sub.2 Chicken II: pGlu--His--Trp--Ser--His--Gly--Trp--Tyr--Pro--Gly--NH.sub.2 __________________________________________________________________________
Chicken I has Gln.sup.8 for Arg.sup.8 of the porcine/ovine LHRH. Chicken II has His.sup.5 for Tyr.sup.5, and Trp.sup.7 Tyr.sup.8 for Leu.sup.7 Arg.sup.8. Salmon LHRH has Trp.sup.7 Leu.sup.8 for Leu.sup.7 Arg.sup.8 of the porcine/ovine LHRH. Chicken II and salmon LHRH have Trp.sup.7, which is an aromatic substitution for the aliphatic Leu.sup.7 of the porcine/ovine LHRH. Each of these four LHRH's may be presumed to have similar but specifically different receptors, but the differences may be so minor that any one of the four receptors might have some significant receptivity to accept any one of the three other LHRH's. The receptor for the porcine/ovine LHRH might effectively accept an antagonist with changes in positions 5, 8 and particularly 7, although Leu.sup.7 is relatively non-functional compared to Trp.sup.7.
Folkers, K., et al. (Biochem. Biophys. Res. Commun. 123(3), 1221-1226 (1984)) synthesized and assayed 13 antagonists having Trp.sup.7 and one of these, [N-Ac-D-2-Nal.sup.1, D-pClPhe.sup.2, D-3-Pal.sup.3, D-Arg.sup.6, Trp.sup.7, D-Ala.sub.10 ]-LHRH not only maintained antagonistic activity, but had increased potency (ca. 90% antiovulatory activity/250 ng; rats) in comparison with a companion analog with the Leu.sup.7 of LHRH (58%).
On the basis of favoring the positive data from several investigators over several years on the existence of FSHRH, rather than the diverse negative data, and assigning special significance to the enhancement of antagonistic activity by having the Trp.sup.7 of the chicken II and salmon LHRH's in place of Leu.sup.7, a new approach was taken toward elucidation of the existence or not of FSHRH.
This new approach was as follows. Of the four known LHRH's, the differences are in positions 5, 7 and 8. His or Tyr occur in position 5, Leu or Trp occur in position 7, and Arg, Leu, Tyr and Gln occur in position 8 for a total involvment of six individual amino acids.
One possibility for the structure of the presumed FSHRH would be that it is a decapeptide with one or more of the same individual six amino acids, and in the same relative positions 5, 7 and 8. Upon this basis, there are a total of 16 possible peptides of which four are the recognized LHRH's. The remaining 12 peptides are as follows:
making only one change:
1. p-Glu His Trp Ser His Gly Leu Arg Pro Gly-NH.sub.2 PA1 2. p-Glu His Trp Ser Tyr Gly Trp Arg Pro Gly-NH.sub.2 PA1 3. p-Glu His Trp Ser Tyr Gly Leu Leu Pro Gly-NH.sub.2 PA1 4. p-Glu His Trp Ser Tyr Gly Leu Tyr Pro Gly-NH.sub.2 PA1 5. p-Glu His Trp Ser His Gly Leu Leu Pro Gly-NH.sub.2 PA1 6. p-Glu His Trp Ser His Gly Leu Tyr Pro Gly-NH.sub.2 PA1 7. p-Glu His Trp Ser His Gly Leu Gln Pro Gly-NH.sub.2 PA1 8. p-Glu His Trp Ser Tyr Gly Trp Tyr Pro Gly-NH.sub.2 PA1 9. p-Glu His Trp Ser Tyr Gly Trp Gln Pro Gly-NH.sub.2 PA1 10. p-Glu His Trp Ser His Gly Trp Arg Pro Gly-NH.sub.2 PA1 11. p-Glu His Trp Ser His Gly Trp Leu Pro Gly-NH.sub.2 PA1 12. p-Glu His Trp Ser His Gly Trp Gln Pro Gly-NH.sub.2
making two changes:
making three changes:
Two of these 12 peptides, numbers 2 and 3, are known, Cone, B. S., et al. (Biochem. Biophys. Res. Commun. 90, 1249 (1979)) and Yanaihara, N. et al. (Biochem. Biophys. Res. Commun. 49(5), 1280 (1972)), respectively. The remaining 10 peptides have been newly synthesized and the [Trp.sup.7 ]-LHRH has been resynthesized, and all of them were assayed in comparison with LHRH. The results are described herein.
We had considered that perhaps one of these 12 peptides might have remarkable hormonal activity in comparision with that of LHRH. Two of the 12 were considered to be unlikely to have biological interest, but there was no way to predict that even one of the remaining 10 would be biologically important, but it was considered that if just one were important on testing, such a result could be an important discovery. Unexpectedly, not only one, but two were discovered to be superior in potency and activity to LHRH, and three more, for a total of five, were comparable in activity to LHRH. We report the results of these syntheses and bioassays and state the diverse utilities herein.